Protein Kinases and Protein Kinase Inhibitors
Protein kinases (PKs) play a key role in the regulation of protein functions in living cells. It has been estimated that the activity of one third of proteins is regulated through phosphorylation of one or more of serine, theorine and thyrosine residues of the protein. More than 400 human diseases (incl. cancer) have been linked to aberrant protein kinase signaling. This has made PK the second largest drug target (and fastest growing category of drugs in development) after G protein-coupled receptors. [Cohen, Nat. Rev. Drug Discov. 1 (2002) 309; Fischer, Curr. Med. Chem. 11 (2004) 1563]
Protein kinases follow ternary complex kinetic mechanism in which direct transfer of the phosphoryl group from ATP to the protein substrate occurs in the active site. [Adams, Chem. Rev. 101 (2001) 2271]
Three different kind of active site-targeted protein kinase inhibitors are previously known. Firstly, despite serious selectivity problems (all 500 protein kinases and more than 1500 other proteins are able to bind purine nucleotides), and high concentration of competing ATP in the cellular milieu, the main efforts of drug companies have been directed to the development of ATP competitive inhibitors. Imatinib, a specific small molecule inhibitor of the Abl kinase, has become the first successful breakthrough in kinase-targeted cancer therapy.
The second type of active site targeted inhibitors of protein kinases comprise compounds that selectively interfere with protein-protein interactions and block the binding of the substrate protein to the active site of the protein kinase. [Recent reviews: Bogoyevitch et al., Biochim. Biophys. Acta. 1754 (2005) 79; Lawrence, New Design Strategies for Ligands That Target Protein Kinase-Mediated Protein-Protein Interactions.; Pinna, A. L., Cohen, P. T. W. Eds.; 2005; p. 11]
Thirdly, combination of the aforementioned approaches and development of bisubstrate-analog (biligand) inhibitors that simultaneously associate with both ATP and protein binding domains of the dual substrate enzyme has given selective and potent inhibitors of PK. Several strategies of the design of bisubstrate-analog inhibitors have been described. [For a review on the subject, see: Parang et al., Pharmacol. Therap. 93 (2002) 145]. Construction of bisubstrate-analogue (bifunctional) inhibitors could lead to enhanced specificity and potency in protein kinase inhibition. The inhibitor mimics two natural substrates/ligands and associates with two regions of a given kinase simultaneously. Most bisubstrate analogues have been designed to mimic ATP and the acceptor component. The inhibitors of this kind are covalent conjugates between one moiety inhibiting ATP binding and one moiety inhibiting binding of a protein/peptide substrate to the protein kinase. [Ricouart et al., J. Med. Chem. 34 (1991) 73; Medzihradszky et al., J. Am. Chem. Soc. 116 (1994) 9413; patent application describing bisubstrate analog inhibitors of PK: WO0070029, WO0170770, WO03010281, WO2004110337, EE200300187]
One of the present inventors has previously developed bisubstrate-analog inhibitors for protein serine/threonine kinases PKA and PKC with activities in sub-micromolar region. [Loog et al., Bioorg. Med. Chem. Lett., 9 (1999) 1447, Uri et al., Bioorg. Med. Chem. Lett., 12 (2002) 2117, WO0070029, EE200300187]. These inhibitors comprise moieties of analogs of both substrates of protein kinases: ATP binding site targeted adenosine-5′-carboxylic acid (Adc) and the protein substrate domain directed oligo-(L-arginine). The design of the latter fragment was based on the knowledge that phosphorylation sites of the substrates of basophilic protein kinases (cAPK, PKC, Akt/PKB, PKG, etc.) are flanked by regions rich in arginine or/and lysine residues. [Pinna et al., Biochim. Biophys. Acta. 1314 (1996) 191] Two active fragments of the inhibitors were connected via a tether which length was optimized in structure-activity studies.
After the priority date (Aug. 15, 2006) the synthesis and characterization of bisubstrate-analog inhibitors for protein serine/threonine kinases incorporating D-arginine residues that is the subject of the present patent application was described in two published articles by inventors of the present patent application. [Enkvist et al., J. Med, Chem. 49 (2006) 7150; Viht et al., Anal. Biochem., 362 (2007) 268]
Existing Assay Methods
Development of new assay methods for evaluation of protein kinase inhibitors has run in parallel with the increase of importance of effective inhibitors for drug industry. During recent years, flexible fluorometric kinase assay methods have substituted problematic (e.g., personal risks, environmental hazards, short half-lives of 32P- and 33P-labeled compounds, long exposure times) radiometric methods. Fluorescent methods can be spatially and temporally more focused than radioactive methods, and as such, they are more suitable for application in high-throughput screening (HTS) assays. [Olive, Expert Rev Proteomics. 1 (2004) 327]
Great majority of evaluations of kinase inhibitors is performed in the form of kinetic studies and the new, potential inhibitors are screened and characterized on the basis of their retarding effect on the rate of substrate (peptide or protein) phosphorylation reaction catalyzed by the kinase. In the case of radioactive methods the product of the phosphorylation reaction that is covalently labeled with radioactive phosphor isotope is separated from radioactive ATP and quantified. The application of labor-intensive separation steps and the use of large amounts of radioactivity make these methods problematic for high throughput assays.
Kinetic methods where the amount of the product of the phosphorylation reaction is established by its competition with a fluorescent binder for an antibody (a phosphopeptide formed in a kinase reaction displaces a fluorescently-labeled phosphopeptide from a phospho-specific antibody) or other phosphopeptide binding macromolecule (Immobilized Metal Assay for Phosphochemicals, IMAP) is another class of assays in active use. [e.g., WO9818956]. In this case the change in the fluorescence anisotropy resulting from the displacement of the fluorescent binder from its complex with the antibody is usually measured. Although these methods suit better for HTS assays, they still require an effective substrate, fluorescent binder and high-affinity antibody or other phosphor-binding macromolecule for the method. Large number of measurements has to be performed to characterize the inhibitory compounds with comparable Ki values.
Alternatively, kinase inhibitors may be detected by direct or indirect measurement of the binding of the inhibitor to the kinase. Some small-molecule inhibitors can be conjugated to a fluorescent dye without loosing their binding affinity to kinase. The kinase-bound labeled inhibitor can then be displaced by competitive kinase inhibitors and the change in fluorescent characteristics measured. This interaction therefore forms the basis for a competitive binding assay of kinase inhibitors where no knowledge of the substrate or an antibody to the phosphorylated kinase substrate is required. Several papers and patent applications describe the use of fluorescent probes for determination of the binding characteristics of protein kinase inhibitors. [e.g., Chen et al., 268 (1993) 15812, WO2005/033330]
Limitations of Current Binding Assays
Fluorescent probes with micromolar affinity towards cAPK were disclosed by Chen et al. [Chen et al., 268 (1993) 15812] These ATP-competitive probes have complicated emission characteristics that originate from the fluorescence of both the fluorescent dye and bisindolylmaleimide ligand. Low (micromolar) affinity and complex fluorescence spectrum make it difficult to use these fluorescent conjugates as probes for binding assays. [WO9906590] Other ATP competitive fluorescent probes have been described in literature. [e.g., WO2005/033330]
A fluorescent probe targeted to the protein/peptide substrate binding domain of the cAMP-dependent protein kinase (cAPK), fluorescein-labeled 20 amino acid residues-containing sequence of protein kinase heat stable inhibitor protein PKI (PKI 5-24), was shown to bind to cARK with micromolar affinity (Kd=1.6 μM) in a fluorescence polarization assay. [Shneider et al., Org. Lett. 7 (2005) 1695] Low binding affinity of the probe prevents its application for the determination of the binding constants of the protein/peptide substrate-binding domain targeted inhibitors.
The main limitations of the binding assays that have been disclosed so far are the following:                A. Fluorescent probes available have low affinity (usually micromolar or submicromolar) towards protein kinases that leads to substantially higher consumption of kinases when using these methods if compared to kinetic assays, and to the impossibility of the application of the probes for determination of exact binding characteristics of high-affinity inhibitors;        B. Fluorescent probes available are active to a single kinase or a small family of kinases that makes it impossible to use a single probe for generic characterization of inhibitors with multiple kinases;        C. All fluorescent probes available thus far enable the testing of compounds binding to either ATP or protein/peptide substrate binding site and as such differently from kinetic methods do not permit simultaneous screening of ATP and protein-peptide substrate binding site-targeted inhibitors.        
To our knowledge, prior art published by other groups most related to the current invention is described in the patent application WO2005/033330 “Fluorescent probes for use in protein kinase inhibitor binding assay”. This invention describes a fluorescent probe with affinity in high nanomolar range (Kd=161 nM) which leads to the need for high concentration of the kinase in the assay format (200 nM STK12 kinase was used in the disclosed Example). Requested kinase concentration, arising from the Kd value of the fluorescent probe, is a hundred-fold higher than the kinase concentration usually applied for kinetic measurements (ca 1 nM concentration of the enzyme is often used in kinetic assays). This leads to substantial increase of the cost of the assay. Due to sub-micromolar dissociation constant of the probe it cannot be used for determination of binding constants of inhibitors with nanomolar affinity [Fluorescence Polarization Technical Resource Guide THIRD EDITION 2004, Invitrogen Corporation. Chapter 7]
The probe disclosed in WO2005/033330 is targeted to the ATP binding site of the kinase and as such the application of the probe for screening of PK inhibitors neglects the inhibitors targeted to the protein/peptide binding domain of the kinase. Furthermore, the fluorescent probe disclosed in WO2005/033330 is applicable for testing of inhibitors of a specific kinase (STK12 and close analogs).
Benefits of the Presented Invention
The invention described in current patent application overcomes limitations of all known fluorescent probes and is applicable in competitive displacement assays for identification and characterization of inhibitors of many protein kinases and determination of the concentration of the protein kinase. If compared to the probes from previous inventions, the Fluorescent probe of the present invention has very high affinity (Kd≦1 nM) and can be used for testing of inhibitors of a variety of protein kinases targeted to both ATP and/or protein substrate binding sites.